Abstract

   We report the proteomic profile of Epidermal Mucus (EM) from Labeo
   rohita and identified the differentially abundant proteins (DAPs)
   against Aeromonas hydrophila infection through label-free liquid
   chromatography-mass spectrometry (LC-MS/MS). Using discovery-based
   proteomics, a total of 2039 proteins were quantified in nontreated
   group and 1,328 proteins in the treated group, of which 114 were
   identified as DAPs in both the groups. Of the 114 DAPs, 68 proteins
   were upregulated and 46 proteins were downregulated in the treated
   group compared to nontreated group. Functional annotations of these
   DAPs shows their association with metabolism, cellular process,
   molecular process, cytoskeletal, stress, and particularly immune
   system. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment
   analysis and Fisher's exact test between the two groups shows that most
   of the proteins were immune-related, which were significantly
   associated with the proteasome, phagosome, and Salmonella infection
   pathways. Overall, this study shows a basic and primary way for further
   functional research of the involvement of vitellogenin 2,
   alpha-2-macroglobulin-like protein, toll-like receptors (TLR-13),
   calpain, keratin-like proteins, and heat shock proteins against
   bacterial infection. Nonetheless, this first-ever comprehensive report
   of a proteomic sketch of EM from L. rohita after A. hydrophila
   infection provides systematic protein information to broadly understand
   the biological role of fish EM against bacterial infection.

Introduction

   Bacterial diseases are one of the important cause of mortality in
   freshwater fish that result in a huge economic loss [[35]27,[36]64]. A
   number of bacteria that cause disease in almost all fish and every
   culture condition, has been reported and need to be focused
   [[37]5,[38]70]. Aeromonas hydrophila is one of the most harmful
   bacteria in almost all animal taxa, and causes septicemia and
   ulceration in freshwater fish [[39]11,[40]33]. A. hydrophila enters
   fish body through the skin and gills [41][13], stimulating macrophage
   apoptosis and destroying the mucus layer [42][59]. This bacterium also
   affects various internal organs such as the liver, kidneys, and spleen
   [43][35]. Contrarly, fish is equipped with many immune factors that
   confer protection against A. hydrophila [[44]74,[45]76]. Particulalry,
   fish epidermal mucus (EM) is proven to protect the host body against
   bacterial infection [[46]21,[47]49].

   The fish EM mucus is multifunctional with a role in respirations,
   osmeoregulation and homeostasis. The composition of fish EM vary across
   the body surface and species to species [[48]3,[49]57]. It contain
   immune relevant components with a significant role in both innate and
   adaptive immunity [50][71]. The structural part of the fish EM is
   mainly composed of glycoprotein (Mucins) [[51]14,[52]18,[53]47,[54]48]
   lipid, ions, enzymes, other immune-related components such as lysozyme
   [[55]28,[56]44], immunoglobulin [[57]7,[58]53,[59]71], lectins
   [60][47], complement [61][1], antimicrobial peptides [[62]26,[63]73],
   proteases [[64]8,[65]66], and heat shock proteins [66][46]. Though
   studies of fish mucosal immunity at protein level have been conducted
   [[67]50,[68]68] yet study of host immune factors in fish has been
   further recommended [[69]15,[70]16].

   In Pakistan, freshwater fish are widely cultivated, though the fish
   industry of this country is not well developed. In addition, pathogenic
   bacteria have been reported in fish [[71]60,[72]69]. Previously, we
   have confirmed several pathogenic bacteria from diseased fish
   inhabiting in different farms [73][2]. Consequently, we found that skin
   mucus from both infected and healthy fish, from the aquatic environment
   of Pakistan, exhibit strong bactericidal activity against different
   kinds of bacteria [[74]2,[75]4]. However, studies regarding fish immune
   system around this country are still very rare. Therefore, we have
   established the proteomic profile of EM isolated from L. rohita treated
   with A. hydrophila. The DAPs we discovered from EM are known for their
   immune functions against bacteria. This basic report will pave new ways
   for further studies and will add important understandings to the local
   and global field of molecular immunology of fish.

Materials and methods

Fish collection

   Fish with an average weight of 799 ± 0.577 g was collected from the
   local fish farm of district Kohat and brought to the Fisheries and
   Aquaculture Laboratory, Department of Zoology, KUST, Kohat. Before
   bacterial infections, 36 fish were distributed in 6 rectangular glass
   aquaria (each with 6 fish) with 200-liter capacity and fed with
   commercial pelleted feed. Three aquaria were considered as control
   whereas three aquaria were considered as treated group. Fish were
   acclimatized for two weeks, with water temperature ranged from
   26–28 °C, and feces were removed daily and 30 % of tank water was
   renewed on each alternate day. All these experimental procedures were
   permitted by the Ethical Research Committee of KUST wide letter number
   “KUST/Ethical Committee/1447”.

Collection of EM and A. hydrophila challenge

   The median lethal dose (LD50) of A.hydrophila ([76]ON920871) (that was
   isolated and identified from naturally infected fish [77][2] was
   determined as described earlier [78][37], cultured in Tryptic Soy Broth
   (TSB) and incubated overnight at 37 °C. For the treated group, each of
   fish was treated with A.hydrophila at amount of 100 μL of around
   1.5 × 10^9 CFU/mL intra-peritoneally, whereas each of nontreated fish
   (control group) were injected with an equal volume of phosphate buffer
   saline (PBS). Skin lesions were examined in the bacterial-treated group
   of fish after 48 hours while these symptoms were not found in the
   nontreated group. Fish were kept starved for one day after 15- days of
   acclimatization to maximize the secretion of mucus and prevent
   defecation during the process of mucus collection. Fish were
   anesthetized with tricaine Methane sulfonate (MS-222) then placed on a
   sterile tray, cleaned with phosphate buffer saline solution, and
   gradually scraped with a sterile slide from lateral sides. Mucus was
   gently collected without creating any dent on fish surface and to
   prevent fecal contamination. Mucus was collected from both control
   (three aquaria each with 6 fish) and treated (three aquaria each with 6
   fish) groups and labeled as NT-LR 1-3 and T-LR 1-3 respectively. The
   collected mucus was put into falcon tubes (15 mL) and then were sent to
   Malaysia Genome and Vaccine Institute (MGVI) Jalan Bangi, 43000 Kajang,
   Selangor, Malaysia for lyophilization and further process.

Protein extraction and quantifications

   Proteins were extracted from preserved and lypholized mucus samples by
   following the protocol of [[79]42,[80]67,[81]72] with little changes.
   Epidermal mucus was melted on dry ice and diluted with 1000 µL PBS.
   Samples were sonicated (2 × 5 s) with an ultrasonic device (SONICS
   Vibracell VCX750, USA) and centrifuged at 4 °C for 30 min at 15,000 g
   and then the supernatant was collected. A 10 % w/v mixture of
   trichloroacetic acid (TCA) and 0.1 % DL-Dithiothreitol, Sigma, USA
   (DTT) was added to the collected supernatant and incubated at -20 °C
   for 120 min. The TCA and DTT mixture containing mucus samples were
   centrifuged at 10000 g for thirty min at 4 °C. The pellet was
   redissolved in chilled acetone containing 0.1 % DTT and incubated at
   -20 °C for 60 min. The mucus sample was again centrifuged at 10,000 g
   for 30 min, at 4 °C, and the obtained pellet was air-dried for 2–3 min
   and suspended in rehydration buffer (9.8 M urea, 20 mM DTT). The same
   protein extraction procedure was used for both treated and nontreated
   groups. After extraction, protein content was calculated with standard
   Bovine Serum Albumin (BSA), and a protein curve was made by using a
   colorimetric Bradford assay. SDS-PAGE was performed and each protein
   was extracted by the method of [82][32] with little modifications. The
   protein samples were put on a 5 % stacking gel at 75V and a 12 %
   resolving gel at 125V. The gel was stained by using Coomassie Brilliant
   Blue (CBB) staining protocol and heated for 1 minute in the microwave
   oven. Then, the gel was kept at room temperature on a shaker for 15 min
   before being destained with a destaining solution (10 % Acetic acid,
   20 % Methanol, 70 % distal water). For visualization of gel, a Bio-Rad
   GS800 calibrated densitometer (Bio-Rad Laboratories, Hercules, CA, USA)
   with a 32-bit pixel depth and 600-dpi resolution were used
   ([83]Fig. 1).

Fig. 1.

   [84]Fig 1
   [85]Open in a new tab

   SDS-PAGE showed the separation of proteins extracted from all samples
   of EM of L. rohita of nontreated NT-LR 1-3 EM and treated T-LR 1-3 with
   A. hydrophila: Lane 1 (N-T1), Lane 2 (N-T2), Lane 3 (N-T3), Lane 4
   (T1), Lane-5 (T2), Lane-6 (T6). Smobio Enhanced 3-colour High Range
   Protein Marker (5-245 kDa) was used as a standard. Approximately 10 μg
   protein was loaded/lane.

Protein analysis and trypsin digestion

   The aggregated protein bands were cut from the stacking gel after
   running 15 min of SDS-PAGE and the sliced gel bands of size 1mm × 1mm,
   were kept in a clean microcentrifuge tube as described earlier
   [86][19]. For destaining, 100 µL of 50 % acetonitrile (ACN) in 50 mM
   ammonium bicarbonate (ABC) was transferred into the tube with excised
   gel and thus the Coomassie staining was washed off. The tube was vortex
   and shaken for 15 min and this step was repeated 3 times until the gel
   became destained. After destaining, proteins were reduced with 300 µL
   of 10 mM DTT in 100 mM ABC into the tube with excised gel and incubated
   for 30 min at 60 °C. After reduction, proteins were alkylated with
   300 µL of 55 mM indole acetic acid (IAA) in 100 mM ABC into the tube
   with excised gel and incubated for 20 min at room temperature in the
   dark. The reduced and alkylated gel was washed with 1000 µL of 50 % ACN
   in 100 mM ABC for 20 min and 100 µL of 100  % ACN was added and vortex
   for 15 min. Without disposing of the excess solution, the gel was
   dehydrated for 60 min at room temperature in a SpeedVac. The gel was
   rehydrated with 50 µL of 6 ng/µL trypsin in 50 mM ABC along with 50 µL
   of 50 mM ABC. The trypsin digested samples were vortexed after
   overnight incubation at 37 °C and then 100 µL of 100 % ACN was added.
   The liquid was collected in a separate clean tube, add 100 µL of 50 %
   ACN, stirred for 15 min. The digested samples were dried completely
   under SpeedVac and then stored at -20 °C for further analysis.

LC-MS/MS analysis

   A microgram of the sample peptide was subjected after quantifications
   into the column and thus LC-MS/MS was run with a LC gradient of 120 min
   from all the samples. Data were attained through the Orbitrap fusion
   tribrid Mass Spectrometer that was linked with an Easy-nLC Nano Liquid
   Chromatographer (system 1200). Then the samplas subjected to and
   EASY-spray column Acclaim pep map TM C18 100 A^0, 2 µm particle size,
   50 µm id x 15 cm at a flow rate of 5  µL/minute. Peptides were
   dissolved in an Acclaim spray column (50 µm × 15 cm, 2 µm particles,
   pore size of 100 Angstrom (Thermo Fisher Scientific), with a flow rate
   of 250 nL/minute for 120 min and meanwhile the gradient was used as
   solvent B (Acetonitrile with 0.1 percent Formic acid). Spectrometric
   acquisition was done through the Orbitrap Mass analyzer (Data Dependent
   Acquisition mode) with complete scan ranged (310–1800 m/z) where the
   Mass Resolution was 60000. The dynamic exclusion time was set at 20 s
   and the mas window was set at 10 ppm. Collision-Induced Dissociation
   (CID) and High-Energy Collision Dissociation (HCD) methods were used
   for fragmentation at the MS1 and MS2 levels, with AGC targets set at
   400000 and 10000, respectively, to acquire all MS/MS spectra.
   Meanwhile, for positive internal calibration, a lock mass of 445.12003
   m/z was used.

Label free quantifications and identification of proteins (Proteome
Discoverer Version 2.1)

   Integrated search engine in NCBI and Thermo Scientific TM Proteome
   Discoverer Version 2.1 was used to analyse the raw MS data against the
   L. rohita protein database (taxonomy ID - 84645, retrieved on
   09/27/2022). In Proteome Discoverer, the mode of Orbitrap fusion was
   chosen as the tool where trypsin was selected as the protease. Overall,
   2- missed cleavages were permitted as oxidation on methionine selected
   as the variable modification and carbamidomethylation on cysteine as
   the inert alteration. All peptides were validated by using the
   percolator® algorithm, based on a q-value smaller than 1 % False
   Discovery Rate (FDR). The reverse mode was elected as the decoy mode,
   and proteins were detected based exclusively on their unique peptide.

Statistical analysis

   Data obtained with Proteome Discoverer Software were subsequently
   analyzed with the Perseus framework, accessed on 15-10-2022. Utilizing
   the Proteome Discoverer output files, statistical analysis was
   performed by Perseus software (version 2.0.7.0) for the identification
   of DAPs between the two groups. Statistical assessment was executed in
   R programming language to differentiate the DAPs between treated and
   nontreated EM samples. The logarithmic values and missing data were
   overwritten by imputing missing values following the normal
   distribution. Then, the rows were filtered for valid values that
   contained at least 3 values in one group. All missing values were
   replaced by a normal distribution using the imputation function. To
   obtain an estimate of the variabilities among the biological replicates
   of the nontreated and treated, DE analysis (RStudio) was applied.

Functional annotation

   Selected differentially abundant proteins (DAPs) from the Perseus data
   were used for functional annotation and biological pathway analysis.
   The DAPs identified from Perseus and the NCBI database were integrated
   into Ubuntu to obtain the sequence for each accession and then
   submitted to Euro-galaxy to blast the sequences. Subsequently, the
   blastp.xml files and FASTA files retrieved from Ubuntu were used for
   functional annotations by using Blast2GO and OMICSBOX. Enrichment
   analysis (Fisher's exact test) was performed for all DAPs (upregulated
   v downregulated proteins) (P ≤ 0.05).

Results

A. hydrophila infection alters proteomic profile of L. rohita EM

   Discovery-based proteomics data by high-resolution MS identified and
   quantified the proteins in the EM. The significantly unique peptides
   (P ≤ 0.05) were used to identify the group of proteins that
   differentiate the treated and nontreated groups, where comparison was
   performed using nontreated group as a reference group. A sum of 2039 in
   nontreated (NT-LR-1 (2932), NT-LR-2 (1516), NT-LR-3 (1669), and 1,328
   proteins were found in the treated groups T-LR-1 (1365), T-LR-2 (1102),
   T-LR-3 (1519). Furthermore, a total of 114 proteins were found as DAPs
   after Perseus analysis between the two groups (NT-LR v T-LR). Of these
   114 DAPs, 46 were downregulated and 68 were upregulated in the treated
   group compared to nontreated group. The plots of all the measured
   upregulated and downregulated proteins along with the fold changes are
   shown in [87]Table 1.

Table 1.

   Differentially abundant proteins (DAPs) found between treated and
   nontreated Epidermal mucus of L. rohita samples
   Accession Protein Name Coverage[88]^a Sequence score[89]^b Molecular
   weight[90]^c P-value[91]^d Fold change logFC[92]^e Peptides[93]^f
   UPREGULATED PROTEINS
   [94]KAI2654466.1 14-3-3 protein zeta/delta 32.24489796 356.0382429
   27.83 1.22E-19 22.91038174 7
   [95]RXN25852.1 transferrin variant C 6.433824 118.9949 60.348 0.002643
   21.8081482 3
   [96]RXN12930.1 WD repeat-containing 34 3.225806452 51.80760956 82.672
   0.008918 21.78980799 2
   [97]RXN05447.1 toll-like receptor 13 3.067484663 10.5339762 74.829
   0.004373 21.6107732 2
   [98]RXN03646.1 type I cytoskeletal 19-like protein 7.136715391
   147.1744276 127.673 0.008918 21.47489227 5
   [99]RXN39545.1 apolipo A-I 11.95652174 102.0174513 31.868 4.35E-20
   21.43188226 3
   [100]RXN02394.1 type I cytoskeletal 18 4.147465438 19.3421793 48.967
   0.004373 21.17485778 2
   [101]RXN13896.1 GTP-binding nuclear Ran 7.726269316 55.95985115 50.788
   0.038607 20.95849278 3
   [102]RXN32036.1 annexin A1-like protein 10.52631579 141.2285335 37.693
   0.007678 20.8807011 3
   [103]RXN34607.1 type I cytoskeletal 13-like protein 14.25178147
   143.731734 46.866 0.028555 20.85871854 6
   [104]RXN28728.1 calpain small subunit 1-like protein 12.03703704
   9.394081831 24.41 0.009598 20.80147232 2
   [105]RXN28728.1 calpain small subunit 1-like protein 12.03703704
   9.394081831 24.41 0.009598 20.80147232 2
   [106]RXN24735.1 coactosin 31.69014085 85.82882822 16.084 0.028555
   20.79489129 3
   [107]KAI2649895.1 Keratin, type II cytoskeletal 8 12.09540034
   424.1445801 64.975 0.014761 20.43275733 7
   [108]RXN29259.1 tubulin beta-4B chain 21.7847769 496.9043911 84.759
   0.007678 20.42212607 14
   [109]RXN22594.1 AP-2 complex subunit beta isoform X2 4.124860647
   43.48040962 99.817 0.038607 20.35167945 3
   [110]KAI2659941.1 14-3-3 protein gamma 21.95121951 172.9431078 27.915
   0.028555 20.21073983 5
   [111]RXN10255.1 14 kDa apolipo 16.25441696 133.5789763 31.576 0.000432
   20.11850056 3
   [112]KAI2657905.1 Phosphoglycerate mutase 1 18.11023622 60.80386984
   28.908 0.004373 20.08165743 4
   [113]RXN34193.1 heterogeneous nuclear ribonucleo A B-like isoform X2
   6.707317073 62.40562987 36.374 0.004373 20.06275069 2
   [114]KAI2664700.1 Upstream stimulatory factor 1 4.081632653 36.81055641
   54.029 0.000432 19.92284092 2
   [115]KAI2648171.1 L-lactate dehydrogenase A chain 17.22222222
   282.8044053 39.534 0.028555 19.91698552 5
   [116]RXN24522.1 proliferating cell nuclear antigen 12.30769231
   6.560997844 28.622 0.011435 19.58838676 2
   [117]KAI2647796.1 Epidermal differentiation-specific protein
   14.53488372 56.28749657 19.287 0.011435 19.58555809 2
   [118]RXN12433.1 profilin-2-like isoform X2 26.61870504 195.6835451
   15.092 0.024958 19.51813189 4
   [119]RXN18447.1 F-actin-capping subunit alpha-1-like protein
   15.01831502 36.7423625 31.309 3.89E-22 19.51565971 2
   [120]RXN05295.1 iron-responsive element-binding 2 1.565074135
   45.93264389 133.37 0.007678 19.36186956 2
   [121]RXN31617.1 calcium calmodulin-dependent kinase type II delta 1
   chain isoform X4 9.241706161 55.17066252 48.085 0.004373 19.34781695 3
   [122]ACJ53925.1 Annexin A4 14.64174455 40.63251173 35.56 0.007678
   19.3164431 3
   [123]RXN07412.1 ADP ATP translocase 2 protein heart- 2.258355917
   26.85654998 123.753 0.000425 19.26803822 2
   [124]KAI2659359.1 Actin-related protein 2/3 complex subunit 4
   13.21585903 42.43894362 26.366 0.011435 19.25705307 3
   [125]KAI2660878.1 Keratin, type I cytoskeletal 18 2.11673614 0.066 549
   6.75E-23 19.2502566 1.91925991
   [126]RXN20411.1 ras-related Rab-11B 15.59633028 33.24337554 24.524
   0.007678 19.23104757 3
   [127]RXN22617.1 gelsolin-like isoform X2 8.923512748 102.4767544 77.409
   0.002643 19.16384178 3
   [128]KAG7252158.1 hypothetical protein CRUP_019583 10.45454545
   62.98877025 25.631 0.002643 19.06175059 2
   [129]RXN23302.1 F-actin-capping subunit 29.28870293 122.9185866 26.827
   0.007678 18.93273531 5
   [130]KAI2652355.1 Heat shock protein HSP 90-alpha 1 14.69387755
   307.9437141 168.849 0.025211 18.89629376 10
   [131]RXN09298.1 lysozyme g-like protein 11.73469388 59.77187526 21.751
   0.008215 18.79699104 2
   [132]RXN23886.1 tubulin alpha chain 33.33333333 773.8345332 65.228
   0.004373 18.7601335 9
   [133]RXN03073.1 40S ribosomal S12 18.18181818 14.07801127 14.457
   0.008918 18.74137322 2
   [134]KAI2668313.1 NADH dehydrogenase [ubiquinone] iron-sulfur protein
   8, mitochondrial 10.95238095 11.1543324 23.722 1.42E-27 18.64521205 2
   [135]RXN34167.1 Peptide-N4-N-acetyl-beta-D-glucosaminylasparagine
   amidase F precursor 1.968911917 37.69668472 109.2 0.007678 18.55714769
   2
   [136]RXN33499.1 carboxylesterase notum2-like protein 2.812148481
   38.19678736 99.267 0.038607 18.50430993 2
   [137]RXN15989.1 annexin A4 13.75892287 0.445 35.662 1.26E-29
   18.49957111 4
   [138]KAI2664763.1 Annexin A1 4.517704518 25.98113525 89.231 0.001942
   18.43592754 3
   [139]RXN33734.1 E3 ubiquitin- ligase TRIM32 5.461768 0.166 1007
   0.002643 18.43159015 89.88571
   [140]RXN25902.1 complement C3-like protein 2.873563 107.5233 175.633
   0.007678 18.26903485 4
   [141]RXN12299.1 40S ribosomal S3a 16.10486891 75.73952174 30.196
   0.009598 18.24984963 4
   [142]RXN23916.1 alpha-2-macroglobulin-like protein 5.094112 205.2019
   645.484 0.004373 18.19080816 7
   [143]RXN23912.1 alpha-2-macroglobulin-like protein 9.89011 0.432 1274
   1.01E-27 18.17669055 572.9518
   [144]RXN31849.1 26S proteasome non-ATPase regulatory subunit 7 7.317073
   52.61989 37.211 0.008918 17.83203548 4
   [145]RXN19027.1 actin-related 2-like protein 7.868020305 84.86260021
   44.307 7.67E-37 17.72308091 2
   [146]AUG68890.1 RAB7 17.87439614 114.1709089 23.538 0.008918
   17.68621479 3
   [147]KAI2657013.1 Macrophage migration inhibitory factor 26.95652174
   16.91188049 12.371 0.008918 17.58408073 2
   [148]APB93353.1 vitellogenin 2 4.679802956 125.8058792 178.33 0.011435
   17.57444871 7
   [149]RXN07670.1 calpain small subunit 1-like protein 10.56910569
   94.73768556 41.925 0.014761 17.51999958 3
   [150]RXN23913.1 alpha-2-macroglobulin-like protein 8.767577 99.81071
   135.223 0.007678 17.41664103 6
   [151]KAI2646821.1 Histone H4 25.29833 868.5344 46.687 0.004373 4.552064
   13
   [152]AVP32215.1 14-3-3 protein beta/alpha-A 42.21311 420.4139 27.616
   0.007678 3.797519 7
   [153]KAI2653804.1 Galectin-4 24.80276 430.8551 225.596 0.008918
   3.676603 7
   [154]KAI2655256.1 14-3-3 protein beta/alpha-A 6.811146 109.3536 36.085
   0.011435 3.235041 2
   [155]RXN10339.1 60S ribosomal L12 isoform X1 23.39181 159.9536 18.361
   0.004373 3.222491 3
   [156]RXN05230.1 type I cytoskeletal 13-like protein 22.34568 1008.685
   89.213 0.007678 2.973874 13
   [157]RXN24768.1 epithelial cell adhesion molecule-like protein 5.096661
   82.86241 64.769 0.028555 2.967842 2
   [158]KAI2666578.1 40S ribosomal protein S2 7.865169 78.21855 48.399
   0.038607 2.962757 3
   [159]KAI2655262.1 40S ribosomal protein S19 13.42282 64.83232 16.334
   0.008918 2.871513 2
   [160]RXN02566.1 cytolysin Src-1-like protein 9.803922 109.0125 22.992
   0.002643 2.737077 2
   DOWNREGULATED PROTEINS
   [161]RXN14569.1 type I cytoskeletal 47 kDa-like isoform X1 7.129094
   71.91799 54.307 0.011435 -4.549691 4
   [162]KAI2645748.1 Caspase-6 5.333333 0.122 300 0.004373 -17.24014683
   9.682151
   [163]KAI2664457.1 Vitamin D-binding protein 2.591793 0.155 463 0.004373
   -17.42566514 16.42327
   [164]KAI2665451.1 Cullin-associated NEDD8-dissociated protein 1
   1.572739 40.56822 169.257 3.27E-34 -17.49760959 2
   [165]KAI2662642.1 Cysteine–tRNA ligase, cytoplasmic 18.98 6.166388
   93.666 0.007678 -17.50674528 2
   [166]KAI2668644.1 Phosphoglucomutase-2 4.746318 0.059 611 0.011435
   -17.54393459 24.30084
   [167]RXN18438.1 kelch 12 15 471.1007 111.734 0.008918 -17.89792701 6
   [168]RXN27388.1 calpain-2 catalytic subunit-like protein 3.555556 0.062
   675 0.004373 -17.91502237 13.33443
   [169]RXN14036.1 cytochrome b-c1 complex subunit mitochondrial-like
   protein 3.043478 0.28 460 0.007678 -18.03000664 43.02423
   [170]AFY09726.1 ceruloplasmin, partial 2.299908 0.041 1087 0.007678
   -18.05918035 9.313047
   [171]KAI2651062.1 Fructose-bisphosphate aldolase C 25.89532 0.551 363
   0.008918 -18.20300257 64.69082
   [172]KAI2665055.1 Cystatin-B 15 0.292 100 0.008918 -18.23523105
   34.78967
   [173]RXN13828.1 complement C3-like protein 3.011803 45.17195 274.04
   4.49E-27 -18.23523105 2
   [174]RXN13544.1 guanine nucleotide-binding G(I) G(S) G(T) subunit
   beta-1 1.84667 64.88922 199.371 1.14E-26 -18.27436767 4
   [175]RXN18980.1 alpha-2-macroglobulin-like protein 4.251208 53.82425
   115.324 0.038607 -18.31396833 3
   [176]RXN04861.1 serine threonine- phosphatase 2A catalytic subunit
   alpha isoform 18.08118 0.668 271 3.33E-26 -18.32181989 87.60896
   [177]KAI2664321.1 Heterogeneous nuclear ribonucleoprotein U-like
   protein 1 3.272727 0.099 825 0.007678 -18.32781097 50.25338
   [178]KAI2664776.1 AP-1 complex subunit beta-1 2.495379 18.65408 119.297
   0.011435 -18.34401964 2
   [179]KAI2661976.1 Voltage-dependent anion-selective channel protein 2
   7.389163 0.105 406 0.038607 -18.3863855 12.7973
   [180]RXN20681.1 GC-rich sequence DNA-binding factor 2-like protein
   3.49345 207.1441 104.327 0.007678 -18.45002047 2
   [181]KAI2651791.1 hypothetical protein H4Q32_014542 8.520179 0.369 446
   0.038607 -18.46458762 138.7432
   [182]KAI2663457.1 Envoplakin 0.73107 0.017 1915 0.028555 -18.47001487
   28.23277
   [183]RXN09211.1 vinculin 2.439024 51.58951 116.273 0.011435
   -18.52705845 3
   [184]RXN12839.1 ATP-citrate synthase 11.34884 87.34674 117.863 0.008918
   -18.74670881 5
   [185]RXN26255.1 programmed cell death 6-interacting -like isoform X2
   0.785714 21.48167 155.173 0.004373 -18.87969881 3
   [186]KAI2664610.1 Beta-hexosaminidase subunit beta 4.32526 37.01671
   66.088 0.008918 -18.93798233 4
   [187]KAI2647972.1 Tubulin beta-4B chain 28.15315 170.167 49.585
   0.011435 -18.94326785 10
   [188]RXN26318.1 catenin beta-1 2.5 55.20581 87.574 2.26E-22
   -18.96900941 2
   [189]RXN06610.1 fibrinogen gamma chain-like protein 8.788599 103.2149
   48.172 2.51E-22 -18.9854998 2
   [190]KAI2657647.1 Annexin A11 4.562044 42.94753 57.791 0.028555
   -19.02265132 2
   [191]RXN27779.1 glutamate mitochondrial-like protein 15.38462 0.719 520
   0.008918 -19.04012907 210.2785
   [192]RXN24956.1 stress-70 mitochondrial 6.811594 96.2167 74.697
   0.008918 -19.16444136 7
   [193]RXN23518.1 elongation factor 2 11.30742 196.7013 94.419 0.028555
   -19.1948385 8
   [194]RXN09288.1 fructose-bisphosphate aldolase B 20.32967 0.823 364
   0.008918 -19.34806304 115.8411
   [195]RXN03876.1 dihydrolipoyl mitochondrial 10.43307 36.79569 53.493
   0.002643 -19.3620008 2
   [196]RXN06894.1 clathrin heavy chain 1 6.416465 212.0428 188.344
   0.007678 -19.8346728 8
   [197]RXN02662.1 ubiquitin-like modifier-activating enzyme 1 5.253104
   273.5401 116.745 0.008918 -19.87320864 5
   [198]KAI2667771.1 Coatomer subunit beta' 3.584906 69.11857 119.633
   0.004373 -19.93365687 4
   [199]RXN26576.1 leukocyte elastase inhibitor-like isoform X1 8.702409
   292.963 148.004 2.12E-20 -20.05134713 5
   [200]RXN26308.1 prolyl endopeptidase-like protein 1.849218 167.7424
   79.763 0.028555 -20.08363065 5
   [201]KAI2647051.1 Class I histocompatibility antigen, F10 alpha chain
   5.112474 96.79037 54.663 0.008918 -20.09890655 2
   [202]RXN12794.1 heat shock 70 17.88491 446.8307 70.451 0.004373
   -20.31155099 5
   [203]RXN26579.1 GDP-mannose 4,6 dehydratase 18.88889 114.4027 29.937
   4.70E-20 -20.50182008 3
   [204]AAY43356.2 intelectin 16.03774 100.6411 35.241 0.007678
   -20.66881279 3
   [205]RXN23766.1 plastin-3 20.25518 377.2351 70.129 4.53E-20
   -20.80099656 8
   [206]RXN04579.1 calpain-2 catalytic subunit-like protein 1.525659
   34.32399 80.19 0.028555 -21.63974299 2
   [207]RXN07826.1 gelsolin-like protein 7.494145 277.8087 94.005 0.004373
   -22.49026236 7
   [208]Open in a new tab
   ^a

   Coverage shows the protein sequence
   ^b

   Sequence Score shows identification of protein score
   ^c

   Molecular weight indicates the proteins size
   ^d

   Proteins values considered significant (P ≤ 0.05)
   ^e

   Fold change are measured as ≥1 and ≤−1
   ^f

   Peptide labels peptide number sequences matched a protein.

   ^gThe proteins numbers are calculated: 46 (Downregulated); 68
   (Upregulated) proteins.

Function annotations of DAPs

   We have compared DAPs between treated and nontreated which showed an
   enrichment of specific functions using functional analysis (BLAS2GO).
   Based on the functions they annotated to, the DAPs were further divided
   into 3 categories: A) biological processes, B) molecular functions, and
   C) cellular components. Due to the importance of EM in the processes of
   immunity, inflammation, stress, skin regeneration, and antibacterial
   activity, we found the proteins sequence mainly associated with immune
   relevant, stress, antibacterial and structural proteins. The number of
   proteins in most of the GO terms in the treated and nontreated groups
   was annotated to developmental process (GO: 0032502); regulation of
   biological process(GO: 0048856); metabolic process (GO: 0008152);
   response to stimulus (GO: 0050896); (GO:0005575); Intracellular
   anatomical entity (GO:0110165); Protein containing complex (GO:
   0032991); membranes (GO: 00432246): C): (GO: 0003674); Structural
   molecular activity (GO: 0005515): binding (GO: 0003824) and catalytic
   activity (GO: 0003824). The 5 GO terms annotated with the highest
   number of proteins were the developmental process, biological process,
   metabolic process, response to stimulus, internal anatomy, and
   molecular functions ([209]Fig. 2).

Fig. 2.

   [210]Fig 2
   [211]Open in a new tab

   Gene Ontology annotations of the DAPs of both NT-LR and T-LR groups
   based on the BLAST2GO (P ≤ 0.05). Bar graph is the identified proteins
   enriched with gene ontology (GO) after classification into 3-categories
   on the basis of enrichment score. GO terms: A): Biological Process
   (GO:0065007); developmental process (GO: 0032502); regulation of
   biological process (GO: 0048856); metabolic process (GO: 0008152);
   response to stimulus (GO: 0050896); B): Cellular Component
   (GO:0005575); Intracellular anatomical entity (GO:0110165); Protein
   containing complex (GO: 0032991); membranes (GO: 00432246): C):
   Molecular Function (GO: 0003674); Structural molecular activity (GO:
   0005515): binding (GO: 0003824).

   Functional annotation of EM proteins belonged to specific pathways and
   has an important role in stress, immune, and cytoskeleton and are
   significantly upregulated after treated with A. hydrophila. The notable
   proteins were Ubiquitin-conjugating enzyme (E3 ubiquitin- ligase
   TRIM32) and 26S proteasome non-ATPase regulatory subunit 7 and etcetera
   ([212]Table 1). Many biological processes depend on ubiquitination,
   including regulation of transcription, protein transport, progression
   of the cell cycle, immune response, signal transduction, and
   pathogenesis. Bacterial infections can get the benefit of the fish
   ubiquitin system to use the responses of the immune system for their
   purpose. Proteins as transferrin variant C, type I cytoskeletal 13-like
   protein type I cytoskeletal 18, type I cytoskeletal 19-like protein,
   14-3-3 protein zeta/delta, apolipo A-I, Galectin-4, which are
   significantly upregulated in the EM of treated group. Galectin is a
   lectin binding protein that has increased significantly after exposure
   to A. hydrophila. Intelectin has an important function in clumping
   bacteria and has significant role in the nonspecific immune system
   ([213]Table 1).

Upregulated and Downregulated proteins of L. rohita EM

   Comparison between the treated and nontreated groups discovered DAPs
   against A. hydrophila infection. Functional annotations showed that
   most of the downregulated DAPs involved in developmental processes,
   metabolism process, gene regulation processes, immune related proteins,
   and oxidoreductase activity. While upregulated DAPs were mainly mapped
   to antibacterial proteins, immune related proteins, developmental and
   metabolic processes, cellular metabolism, cytoplasmic components,
   molecular components, carboxylic acid metabolism, and binding proteins
   ([214]Fig. 3).

Fig. 3.

   [215]Fig 3
   [216]Open in a new tab

   Fisher Exact Test (Enrichment analysis): Fisher exact test was
   performed for the comparison of GO terms based on sequence percentage
   (Biological process, Cellular components, and Molecular function) of
   the DAPs (Upregulated v downregulated).

   As shown the list of DAPs, the immune response against A. hydrophila
   upregulates and abound the immune-related protein, viz., annexin
   A1-like protein, apolipo A-I, Galectin-4, epithelial cell adhesion
   molecule-like protein, calpain, keratin like proteins, hypothetical
   protein CRUP_019583, Histone H4, heat shock HSPs 90, Complement C3 like
   protein and TLR-13. Whereas, unlikely heat shock protein 70, Class I
   histocompatibility antigen, F10 alpha chain, hypothetical protein
   H4Q32_014542 were downregulated ([217]Table 1).

Enrichment analysis of DAPs and pathways

   KEGG enrichment pathways analysis of the 114 annotated DAPs was
   involved in the regulation of actin cytoskeleton, calcium signalling
   pathway, NOD like receptor signalling pathways, metabolic pathways,
   phagosome, complement pathways, biosynthesis of amino acids,
   Proteasome, and salmonella infection. Moreover, salmonella infections,
   metabolic pathways, phagosome, and Proteasome pathways were the
   significant identified pathways in the treated EM of L. rohita after
   KEGG analysis ([218]Fig. 4).

Fig. 4.

   [219]Fig 4
   [220]Open in a new tab

   KEGG pathways enrichment analysis for the identified DAPs. Each row
   denotes an enriched function, and the count denotes the enriched ratio
   which is reflected as the "input proteins number". The colour of the
   bar differentiates Up and downregulated pathways.

A. Hydrophila abound the cytoskeleton, lysosomal and apoptotic proteins in L.
rohita EM

   The cytoskeletal proteins including Actin-related protein 2/3 complex
   subunit 4, Keratin, tubulin alpha chain, tubulin beta-4B chain, type I
   cytoskeletal 13-like protein, type I cytoskeletal 18, type I
   cytoskeletal 19-like protein, was found to be upregulated. ATP-binding
   component of the Arp2/3 (Actin-related protein 2/3 complex subunit 4)
   complex maintains the cytoskeletal homeostasis without being treated
   with bacterial infections that aid cell motility by facilitating the
   production of branching actin systems in the cytoplasm. The Arp2/3
   complex protein is essential for lamellipodium protrusion, vesicle
   transferring, and entry of bacteria in the fish cell during exposure.

   Another significant protein, TLR-13 is the recognizer of pathogens,
   found to be upregulated in the EM of treated group ([221]Table 1). TLRs
   recognize molecular pattern pathogen-associated derived from bacteria,
   perform an important role in the maturation and activation of
   macrophages, and C-type lectins are also recognized to boost specific
   immune responses.

Discussion

   This first-ever comprehensive proteomic profile of EM of nontreated and
   bacterial-treated L. rohita reveals unique DAPs and pathways. Pathway
   enrichment analysis and Fisher exact test discovered the contribution
   of these proteins in biological processes, cellular components, and
   molecular functions. Notably, Toll-like receptor-13, Histone H4,
   Galectin-4, complement C3-like proteins, Heat shock proteins HSP90,
   calpain-like small subunit, keratin-like proteins, hypothetical protein
   CRUP_019583 were mapped upregulated proteins. Whereas proteins like
   Class I histocompatibility antigen, F10 alpha chain, hypothetical
   protein H4Q32_014542, were mapped as downregulated proteins.

   In our study, TLR-13 was upregulated in the EM of treated fish
   ([222]Table 1), unlikely it was downregulated in the liver of L. rohita
   treated with A.hydrophila [223][39]. TLRs are the important epidermal
   proteins of innate immune system that acts as a receptor and help in
   the recognition of pathogen-associated molecular patterns (PAMPs)
   [224][40]. TLRs can initiate the TIR-domain comprising adapter-inducing
   interferon-dependent signaling pathway (TRIF) developed by tumor
   necrosis receptor-associated factor, which ultimately activates MAP
   kinase (MEK1/2), NF-κB, or activator protein (AP) [225][69]. TLR-13 is
   generally identified as an endosomal receptor, which is a cell surface
   receptor capable of TRIF-mediated signal transduction in L. rohita
   [226][56]. TLR-13 was the 1^st identified antiviral protein that has
   been described to distinguish dsRNA of several RNA viruses and initiate
   the TIRF pathway, which develops type I interferon (IFN) and
   proinflammatory cytokines [227][29]. Furthermore, TLR-13 has also been
   reported in channel catfish, zebrafish, mice, and yellow croaker to
   work against bacteria and TLR-2 microbial ligand peptidoglycan in
   immature dendritic cells [228][29]. Upregulation of TLR-13 in treated
   Zebrafish with Edwardsiella tarda has been reported at transcriptomic
   level [229][34]. TLR-13 was found to be downregulated in kidney and
   spleen of treated blue catfish [230][6] and rainbow trout with
   Edwardsiella ictulari and Yersinia ruckeri [231][51]. Taking together,
   this altered expression of TLR-13 may be due to the nature of stimulus
   and may be organs and species-specific. In fact, during A. hydrophila
   infection, the infected cell of fish would have tried to remove the
   bacterium by antimicrobial processes, thus interfered with signal
   transduction through the TLR [232][9].

   Calpain small subunit 1-like protein was found to be upregulated in the
   EM of the treated group, whereas calpain-catalytic subunit 2 was
   downregulated in non-treated group ([233]Table 1). Calpain is a
   regulatory component that acts as a calcium-dependent cysteine protease
   and is abundant in mammalian cells [234][65]. Calpain-mediated
   proteolysis is lacking and mostly alters substrate proteins rather than
   complete degradation, suggesting that calpain has a specific role in
   suppressing the physiological response [235][54]. In addition,
   inflammatory cytokines such as TNF-α and interleukins can suppress
   calpain levels in cells and tissues [236][23]. Therefore, the
   inflammatory cytokines induced during exposure of cod fish to Vibrio
   anguillarum may suppress calpain levels [237][48]. In addition,
   epidermal lesions caused by bacteria disrupt the amount of calcium and
   displace the cell membrane, which may affect the stimulation of
   calpain. In our study, no epidermal lesion was found because calpain
   protein was upregulated in the treated group. However, mucosal
   calpains, which are found in higher vertebrates, are thought to play an
   imperative role in mucosal defense [238][55].

   Keratin, type I cytoskeletal-13, 18, and 19 were upregulated in treated
   L. rohita EM ([239]Table 1). Keratin is a cell structural protein whose
   main role is to defend cells from physical and mechanical injury
   [240][55]. Several studies have suggested that keratin present in fish
   epidermal mucus has a bactericidal activity due to its pore-forming
   characteristics [241][36]. The amount of Keratin depends on the
   ubiquitin-proteasome pathway, and its expression levels can be changed
   upon cell damage [242][52]. Keratin II was observed to be downregulated
   in treated Atlantic cod mucus with Vibrio bacteria [243][48].

   Annexins are an important protein of fish EM and have anti-inflammatory
   and antibacterial properties [[244]20,[245]75]. Six different types of
   annexins proteins were reported upregulated in the gills of treated
   channel catfish (i.e., A-1, A-2, A-4, A-5, A-6, and A11) with
   Edwardsiella ictaluri [[246]30,[247]75]. Besides, annexin A-13 can
   persuade a defensive response against the parasite fluke Microcotyle
   sebastis infected the fish gills [248][12]. Annexin A-2 protein was
   significantly increased in the rainbow trout gills by uncovered to X
   rays [249][62]. Annexin max 3 found in medaka (Japanese rice fish) are
   similar to annexin A-2 found in gills of rainbow trout, which showed
   that expression of annexin max 3 protein was upregulated in gills by
   exposure to direct radiation [250][63]. In this study, the annexin A1
   and A4 like protein was upregulated in treated L. rohita, which may be
   indication of an active immune response in epidermal mucus against
   bacteria.

   Histones play an imperative role in the chromatin structure and
   transcriptional regulation and also participate actively in the fish
   immune system [[251]38,[252]58]. Histone proteins such as H2A, H2B, H3,
   and H4 were upregulated in shrimp (Litopenaeus vannamei) in response to
   Micrococcus luteus [253][41]. Histone 4 has bactericidal role in oyster
   (Crassostrea virginica) treated with E. coli, Staphylococcus aureus, V.
   anguillarum, and M. luteus [[254]10,[255]17]. Histone 4 protein was
   also found to be overexpressed in the treated zebrafish gills with A.
   hydrophila, while linker histone like protein H1M was upregulated in EM
   of zebrafish exposed to Citrobacter freundii [256][31]. In our study,
   Histone 4 was upregulated in the EM of treated group signifying that
   histones proteins act as a possible bactericidal agent for the
   prevention and treatment of fish bacterial infection [257][38].

   Beta-tubulin was upregulated in the EM of Gadus morhua treated with V.
   anguillarum suggesting its role in phagocytic activities [258][48].
   Cytoskeleton proteins such as actin and tubulin were identified in the
   gills of zebrafish treated with A. hydrophila possibly has
   immunological role to activate the phagosome pathways. In mammals,
   myosins have an important role in muscle contraction and relaxation
   processes. Tropomyosin, along with the troponin complex, was related to
   actin in muscle fibers and regulates muscle contraction by regulating
   the myosin binding [259][22]. The upregulation of all these proteins
   found in our study suggests that these proteins play an important role
   in the EM of L. rohita against A. hydrophila infection.

   Heat shock proteins (HSPs) are conserved and have role in the stress
   response, counting heat, tissue damage, heavy metal contact, and
   bacterial infection. They are characterized into families based on
   their molecular weight: HSPs (15–30 kDa), HSP60s, HSP70s (66–79 kDa),
   HSP90s (83–90 kDa) and HSP100s. HSPs proteins help in the recovery of
   fish cells in stress conditions and promote cyto-protection
   [[260]43,[261]61]. HSP60 is an inducible stress chaperone that is
   primarily found in the eukaryotic cells' mitochondria and can initiate
   a strong pro-inflammatory response in cells of the fish's innate immune
   system [262][61]. The rapid response of HSP70s to environmental
   stresses in the presence of infections has led to its observation as a
   biomarker [263][25]. Heat shock proteins (HSP90) works also has an
   important role in several activities such as protein degradation, cell
   signaling, and intracellular transport [264][77]. Heat shock proteins
   (HSP) has been found in EM of Dicentrarchus labrax [265][77], Sparus
   aurata [266][26], Cyclopterus lumpus [267][43], and Salmo salar
   [268][46]. In this study, HSP 90-alpha 1 was upregulated in EM of
   treated group and HSP-70 was downregulated ([269]Table 1). This altered
   abundance may indicate their role against A. hydrophila infection and
   immune regulation in L. rohita.

   In our study, the complement protein C3-like protein was upregulated in
   the EM of treated group of L. rohita, while downregulated in the
   nontreated group, suggesting that it may intermediate the recruitment
   of immune cells (chemoattractant), opsonin (stimulation of
   phagocytosis), and agglutinin (coagulation of pathogens) found in the
   skin mucus of Sea bass [270][14]. Apolipo-A1 and 14 kDa Apolipo protein
   were upregulated in the EM of treated group by 21 and 20-fold
   respectively in the present study. Apolipo proteins were upregulated in
   the EM of Atlantic cod and salmon treated with sea lice and bacterial
   infection, respectively [[271]18,[272]48]. Furthermore, Apolipo-1 in
   Striped bass has bactericidal activity in vitro [273][24] and lytic
   activity in the skin mucus of Channel catfish [274][45]. Functional
   diversity of EM is linked to the complex components, and identification
   of DAPs against A. hydrophila might be helpful to understand the
   possible mechanism of EM of L. rohita against bacterial infection. The
   upregulation of EM proteins might be due to due to excess proliferation
   of goblet cell. These results together indicate that A. hydrophila
   infection possibly triggers goblet cells to secret more immune
   components against bacterial infection. Thus, more studies are required
   to determine the function of each described proteins, as well as their
   accurate role and mechanism in the immune response against bacterial
   infection.

Conclusions

   The study presents the comparative proteomic sketches of EM from
   bacterial-treated and nontreated L. rohita. Functional annotations of
   the DAPs show that A. hydrophila treatment may alter the abundance of
   protein in the EM of fish. Most of the DAPs found in this study were
   immune-related, stress-related, bactericidal, and cytoskeletal
   proteins. The significant signalling pathways in which the DAPs were
   involved, were related to Salmonella infection, proteasome, phagosome,
   and metabolism. This study will act as a base for further research
   underpinning the molecular immune response of fish EM against bacterial
   infection.

CRediT authorship contribution statement

   Shandana Ali: Conceptualization, Data curation, Formal analysis,
   Investigation, Methodology. Waheed Ullah: Conceptualization,
   Supervision, Writing – review & editing. Ahmad Faris Seman
   Kamarulzaman: Data curation, Formal analysis. Maizom Hassan: Formal
   analysis. Muhammad Rauf: Investigation, Methodology. Muhammad Nasir
   Khan Khattak: Formal analysis, Investigation, Methodology, Supervision,
   Writing – review & editing. Farman Ullah Dawar: Conceptualization,
   Investigation, Methodology, Supervision, Writing – review & editing.

Declaration of Competing Interest

   The authors declare that they have no known competing financial
   interests or personal relationships that could have appeared to
   influence the work reported in this paper.

Acknowledgments